Amides of methobottromycin

ABSTRACT

Amides of methobottromycin and amethobottromycin and mixtures thereof and processes for preparing these compounds are disclosed. The compounds of this application are active antibiotic agents useful in the treatment of chronic respiratory disease of chickens and infectious sinusitis of turkeys.

United States Patent Wolf et al. Jan. 14, 1975 AMIDES OFMETHOBOTTROMYCIN [56] References Cited [75] Inventors: Frank J. Wolf,Westfield; William J. OTHER PUBLICATIONS Somerset both of Derwent FarmDoc No. 25,374, Abstracting NE [73] Assignee: Merck & Co., Inc., Rahway,NJ. Published [22] Flled: 1972 Primary Examiner-Jerome D. Goldberg [21]Appl. No.: 235,286

Related US. Application Data ABSTRACT [63] Continuation-impart of Ser.No. 7l3,256, March 14, Amides of methobottromycin and amethobottromycinI968, abandoned, which is a continuation-in-part of d i t th f dprocesses f preparing 480,040 these compounds are disclosed. Thecompounds of this application are active antibiotic agents useful in[52] US. Cl 424/121, 424/115, 424/122 the treatment of chronicrespiratory disease of chick [51] Int. Cl A61k 21/00 ens and infectioussinusitis of k [58] Field of Search 424/117, 121,122 4 4 Claims, 2Drawing Figures AMIDES OF METHOBOTTROMYCIN CROSS-REFERENCES TO RELATEDAPPLICATIONS This application is a continuation-in-part of U.S. Ser. No.713,256, filed Mar. 14, 1968, now abandoned, which was acontinuation-in-part of US. Ser. No. 480,040, filed Aug. 16, 1965, nowabandoned.

This invention relates to new antibiotic agents and their use. Moreparticularly, the present invention is concerned with novel, highlyactive antibiotic compounds that are molecular modifications of newcompounds known as methobottromycin and amethobottromycin and mixturesthereof, and their use in the treatment of chronic respiratory diseaseof chickens and infectious sinusitis of turkeys.

The discovery of remarkable antibiotic properties of penicillin andsimilar substances has stimulated great interest in the field ofantibiotic compounds such as: streptomycin, gramicidin, subtilin,bacitracin, chlortetracycline, oxytetracycline, cycloserine, colistin,fervenulin, streptozotocin, novobiocin and the like. In general, suchantibiotics are particularly active against certain gram positivebacteria. Others are active against certain gram negative bacteria andsome are active against both gram negative and gram positive bacteria.However, the activity of these known antibiotics is usually limited to afew pathogenic microorganisms and work has been conducted in this fieldin an attempt to find additional antibiotic substances which would beeffective against other pathogens.

In addition, many bacteria which, at one time, were controlled by knownantibiotics, have developed increasing resistance over the years tothese antibiotic substances. As a result, although some of theseantibiotics have been found to be invaluable in the treatment of variousdiseases, it has been discovered that certain strains of some pathogensdevelop a resistance to various particular antibiotics and,consequently, these antibiotics are no longer active against suchstrains of pathogens or the activity of these antibiotics has beenreduced to such a degree so as to make their use against such pathogensof little consequence.

Many antibiotics appear to be highly active in laboratory tests in vitroand in ova but are destroyed or lose much of their effectiveness whenadministered inside the body, in vivo.

Accordingly, the deficiencies of the known antibiotics have stimulatedfurther research to find other antibiotics which will be highly activeagainst a wider range of pathogens as well as those strains of variousmicroorganisms which are resistant to other antibiotics. This is truenot only with disease-producing bacteria which attack humans but alsofor disease-producing bacteria which attack animals and poultry.

Chronic respiratory disease is a disease of chickens and turkeys, causedby a certain group of microorganisms known as PPLO orpleuropneumonia-like organisms, which have been classified asMycoplasma. This is referred to in the art as PPLO infection. Inchickens the disease may be complicated by a secondary invader, at whichtime the disease is known as chronic respiratory disease complex. Inturkeys this disease appears in two forms. It is called infectioussinusitis when it is in the form that affects the upper respiratorytract, and air sac disease when it affects the lower respiratory areas.For the purposes of simplicity, these diseases will be referred toherein as infectious sinusitis.

In chickens the chronic respiratory diseasesymptoms may be like those ofany other respiratory disease such as Newcastle disease, infectiousbronchitis, laryngotracheitis, fungus infection, etc. The usuallyobserved symptoms are nasal discharge and a slight swelling below theeye. Coughing, sneezing, and a hoarse throat rattle or rale mayaccompany these signs. The symptoms of the disease in turkeys are oftendemonstrated by swollen sinuses with gelatinous exudate, watery eyes andcoughing with chcesey or cloudy air sacs.

The economic loss that accompanies chronic respiratory disease is a dropin egg production by at least 10 to 40 percent, which affects the birdsfor several weeks or months. Poor hatchability of fertile eggs laid byinfected hens can cause additional losses. Mycoplasma (PPLO) causedinfection results in the death of a high percentage of embryos. Loss ofweight in a large percentage of birds is also evident. There is, inaddition, a significant amount of mortality in birds beginning at aboutfour weeks of age.

Infection of birds may occur in a number of ways. Birds may be infectedby contact with other infected birds, usually by an inhalation of nasalexudate from a sneezing bird. In fact, infected chickens or turkeys maybecome sick, and they may become carriers in which they appear to behealthy but are, in fact, infected with pathogenic strains of Mycoplasma(PPLO). In addition, birds may be infected through contaminated litter,manure, water and feed, breeding hens or contaminated hatcheries.Transmission of the disease via the infected embryonated egg contributeslargely to an infected flock.

Chemotherapeutic control of these diseases has been successful with avery limited number of compounds. With one exception, the agents whichhave been found satisfactory are known antibiotics used clinically forother diseases, principally human diseases. The exception is theantibiotic tylosin. Although tylosin is used fairly broadly, strains ofPPLO resistant to it have been encountered, and the antibiotic has beenshown to be toxic in use with turkeys.

Other antibiotics useful for controlling chronic respiratory disease inchickens and infectious sinusitis in turkeys are erythromycin andchlortetracycline or oxytetracycline. However, the dosage levels ofthese antibiotics required to obtain good results are quite high, whichresults in an economic barrier to the user. Other antibiotics known tohave anti-PPLO activity usually require a dosage level too close to thetoxic level to be of practical value. Included in this group areneomycin, kanamycin, and chloramphenicol.

Many other antibacterial antibiotics which are used for other infectionshave been found to be without effect on the PPLO. Examples of thesewould include penicillin and its many derivatives, cycloserine,novobiocin, and many others. As can be seen, this group includes agentswith a wide spectrum of activity, hence their inactivity againstMycoplasma shows how these microbes are a unique and specialized type ofbacterium.

It is an object of the present invention to provide useful antibioticsubstances which are highly effective in controlling the primaryetiologics of the chronic respiratory disease of chickens and infectioussinusitis of turkeys.

An additional object of the present invention is to produce new anduseful antibiotic substances which may be used in higher concentrationsthan those presently available without the resultant danger of toxicity.

Another object of the present invention is to provide antibiotics thathave an acceptable oral absorption for treating chronic respiratorydisease of chickens and infectious sinusitis of turkeys.

A further object of the present invention is to provide antibiotics thatmay be applied in relatively low dosages in the treatment of chronicrespiratory disease of chick ens and infectious sinusitis of turkeys.

Another additional object of the present invention is to provideantibiotics that are active against a wide range of strains ofmycoplasmas, including those belonging to the species M. gallisepticum(PPLO), in the treatment of chronic respiratory disease of chickens andinfectious sinusitis of turkeys.

A still further object of the present invention is to provide antibioticsubstances that not only demonstrate significant antibiotic activity invitro and in ova but also show significant antibiotic activity in vivo.

Another object of the present invention is to provide antibiotics thatdevelop protective antibodies during treatment which prevent laterreinfection.

A still further object of the present invention is to provide a processof preparing these novel antibiotic substance.

Other additional objects of the present invention will become apparentto those skilled in the art by reading the following specification.

The new antibiotic substances of the present invention are produced bymolecular modification of newly discovered species of microorganism. Themicroorganism was isolated from the fermentation broth of a soilactinomycete collected from Canada. This new microorganism has beendesignated Streptomyces canadensis MA-959 in the culture collection ofMerck & Co., Inc., Rahway, New Jersey. A culture thereof has beendeposited with the fermentation section of the American Type CultureCollection, 12301 Parklawn Drive, Rockville, Md., and added to itspermanent culture collection as ATCC 17776. The new products of the newspecies of microorganism, known as methobottromycin andamethobottromycin, are disclosed and claimed in U.S. Pat. Nos. 3,683,073and 3,683,072.

The morphological and cultural characteristics of Streptomycescanadensis MA-959 are set forth in the following:

STREPTOMYCES CANADENSIS MA-959 Morphology Biverticillate. Straightchains of 8-10 spores, a few chains longer. Spores cylindrical (950 XAvg. size 1.0 X 1.7).

Czapek Dox Agar (Sucrose Nitrate) Growth light. Aerial mycelium scant,white. Vegetative growth colorless. Reverse colorless. No solublepigment. Sporulation good.

GlycerolAspargine agar Growth good. Aerial mycelium medium gray.Vegetative growth brown to reddish-brown. Reverse brown toreddish-brown. Soluble pigment brown to reddish-brown (pink in earlystage of growth 1 week). No sporulation observed.

Tomato paste-oatmeal agar growth good. Aerial mycelium medium gray withwhite tufts and pink exudate appearing after 3 weeks. Vegetative growthbrown. Reverse dark brown. Soluble pigment brown. Some sporulation.

Emersons agar Growth moderate. Aerial mycelium scant, light gray.Vegetative growth brown. Reverse brown. Soluble pigment light brown(pink in early stage).

Potato plug Growth good. Colonies smooth, cream to grayish-brown. Aerialmycelium light gray (appears only in drier portion of plug). Solublepigment medium brown (pink in early stage).

Starch agar Growth good. No aerial mycelium. Vegetative growth lightbrown. Reverse light brown. Soluble pigment light brown (pink in earlygrowth stage). Hydrolysis.

Nutrient Gelatin plate Growth good. No aerial mycelium. Vegetativegrowth light brown. Reverse light brown. Soluble pigment light brown.Liquefaction. Gelatin stab Soluble pigment dark greenish-brown.One-third liquefaction.

Calcium Malate agar Growth good. Aerial mycelium scant, pinkish white.Vegetative growth yellowish brown. Slight browning of medium alonggrowth streak.

Tyrosine agar Growth moderate. Aerial mycelium pale pinkish-white.Vegetative growth very light brown. Slight browning of medium.

Peptone-Iron Yeast Extract slant Growth good. No aerial mycelium.Vegetative growth gray. Soluble pigment blue-black at 2 days. Mediumbrown at 3 weeks. Skim Milk agar Growth good. Aerial mycelium pinkishwhite. Vegetative growth light brown. Soluble pigment very light brown.No hydrolysis.

Reduction of nitrates Negative under test conditions in organic andsynthetic media.

Temperature Good growth at 28C. No growth at 50C. Micro-aerophilicgrowth (yeast extract-- -dextrose stab) Heavy surface growth and alongtwothirds of stab line.

Milk Peptronization complete at 3 weeks. No coagulation. Heavy brownishgrowth ring with sparse aerial mycelium (light gray). Soluble pigmentmedium grayish-brown. Alkaline reaction (pH 7.9).

Litmus Milk Peptonization complete in three weeks. No coagulation.Alkaline reaction.

The above description of the microorganismproducing methobottromycin andamethobottromycin is given as illustrative of suitable strains ofStreptomyces which can be used in the production of methobottromycin andamethobottromycin, but it is understood that the information hereindescribed is not to be limited to organisms answering this particulardescription. The present invention also contemplates the use of otherspecies of Streptomyces or mutants of the described organisms such asthose obtained by natural selection or those produced by mutatingagents, for example, x-ray irradiation, ultraviolet irradiation,nitrogen mustards, and the like.

New antibiotics of the present invention are molecular modifications ofthe basic compounds which form salts with acids, both inorganic andorganic, such as hydrochloric, tartaric, salicylic, etc., and othercompounds. The free base forms of methobottromycin and amethobottromycinpossess the following physical and chemical properties:

Methobottromycin a. Crystallizes from ethyl acetate in the form of whiteprisms at a temperature of from about 166 to 167C.

b. Easily soluble in alcohols, esters, ethers, chlorinated solvents andbenzene. c. Partly soluble in water. d. Insoluble in petroleum ether,hexane and the like. e. Has a specific rotation of [a],," in a 5 percentsolution of 95 percent ethanol. Amethobottromycin a. Has a melting pointin the range of from about 154 to about 163C. b. Easily soluble inalcohols, esters, ethers, chlorinated solvents and benzene. c. Partlysoluble in water. d. Insoluble in petroleum ether, hexane and the like.e. Has a specific rotation of [011 in a 5 percent solution of 95 percentethanol. Unfortunately, in contrast to other antibiotics bothmethobottromycin and amethobottromycin are found to be exceedinglycomplex compounds having molecular weights of about 800. Consequently,thus far it has not been possible to determine the exact completecomposition of either methobottromycin or amethobottromycin. It has beenfound that these compositions contain the elements carbon, hydrogen,nitrogen, sulfur and oxygen. The found percentages of these elements areas follows:

Methobottromycin Amethobottromycin C 59.50% C 58.40% H 7.52% H 7.44% N13.50% N 13.42% S 3.90% S 4.77% O 15.58% (by difference) 0 15.97% (bydifference) Total 100.00% Total 100.00%

These data suggest molecular structures C.,,H,, N O S and C ,H N,,O Sfor methobottromycin and amethobottromycin respectively but othersimilar molecular formulae are possible within the experimental error ofthese determinations.

The infrared absorption spectrum of the antibiotics methobottromycin andamethobottromycin in chloroform using sodium chloride prism isillustrated in the accompanying drawings. The more significant of thecharacteristic peaks occur at the following wave lengths expressed inreciprocal centimeters: Methobottromycin 3300, 2950, 1730, 1630-1650,1490,

1360, 1300, 1242, 1160, 1132, 1119, 1102, 980, 806; Amethobottromycin3290, 2980, 1739, 1640-1685,1500,1440,1378,1240,1181,1122,1092,1061,995, 980.

The above infrared spectrum readings can be more clearly seen in theattached drawings.

1n the drawings:

FIG. 1 shows the infrared spectrum of amethobottromycin. FIG. 11 showsthe infrared spectrum of methobottromycin.

Methobottromycin and amethobottromycin exhibit characteristic R; valuesin the following solvent systerns:

Ametho- Methobottrobottromycin mycin n-butyl alcohol saturated with 1%aqueous acetic acid 0.85 085 n-butyl alcohol saturated with 2% aqueouspyridine 0.84 0.84

ethyl acetate saturated with 1% aqueous acetic acid 0.37 0.37

ethyl acetate saturated with 0.1M phosphate buffer (pl-l 7) 0.83 0.83

benzene:hexane:methanol:5% aqueous acetic acid (7:6:10:6) 0.0 0.0

benzene:hexane:methanol:5% aqueous pyridine (7:6:1016) 0.5 0.5

benzene saturated with 1% aqueous acetic acid 0.0 0.0

benzene saturated with 2% aqueous pyridine 0.5 0.5

capryl alcohol saturated with 0.1M: pH 6 phosphate (reverse phase) 0300.19

Characteristics of the antibiotics from which the molecularmodifications of the present invention may also include thin layerchromatography. Thin layer chromatographic plates containing silica gelare developed in 94 percent chloroform and 6 methanol, dried and placedin a chamber containing iodine vapor. A brown stain indicates thepresence of these antibiotics. The R, of the methobottromycin zone is0.64 and the amethobottromycin 'zone is 0.60.

The culture producing methobottromycin and amethobottromycin producesgenerally two types of substances: a netropsin-type antibiotic and abottromycintype antibiotic. The bottromycin group of antibiotics fromwhich methobottromycin and amethobottromycin are extracted, is readilyseparated from the netropsin group by extraction with chloroform fromaqueous solutions. The chloroform extract, after purification, shows thepresence of five antibiotic substances on bioautograph of paper strips.The paper strip system utilized for this test consists of paperimpregnated with capryl alcohol and developed downflow with buffer,wherein the R; of methobottromycin is 0.19 and amethobottromycin is0.30. The five components have been designated components A through E inorder of decreasing polarity. Table A below lists bioactivity of all ofthe components of bottromycin. The first column Staph. MIC is a tubedilution assay which measures the minimal inhibitory concentration ofthe antibiotic in a broth culture of the test microorganism, Mycoplasmagallisepticum (PPLO). The second column In Ova ED is an in ova assaywhich measures the effec- Table A Staph. PPLO Egg Test Component MICug/ml ED ,ug/egg A 3.0 720 B 0.23 13 (Amethobottromycin) C 0.04(Methobottromycin) D 0.16 E 0.23 5

Table B shows that ninhydrin-producing substances are liberated from thefive components on acid hydrolysis and paper chromatography.

percentage present too small to separate As can be seen from Table B,amethobottromycin contains proline but does not contain methyl prolineand methobottromycin does not contain proline but does contain methylproline.

In an aqueous solution at pH greater than 10, these antibiotics areunstable. However, at pH from 3 to 9 these products are stable for 24hours at room temperature.

The molecular modifications of the present invention are derived fromantibiotics which are produced by the aerobic fermentation of Streptomyces canadensis MA- 959 in a suitable aqueous medium. Aqueousmediums such as those employed for the production of other antibioticsare suitable for the production of methobottromycin andamethobottromycin. Such mediums contain sources of carbon and nitrogen,assimilable by the microorganism, and inorganic salts. In addition, thefermentation mediums contain traces of metalnecessary for the growth ofthe microorganism which are usually present in complex sources of carbonand nitrogen in the medium.

In general, carbohydrates such as sugars, for example, dextrose,sucrose, dextrin and the like, are suitable sources of assimilablecarbon. The exact quantity of the carbon source will depend, in part,upon the other ingredients of the medium, but it is usually found thatan amount of carbohydrate between about 1 and 6 percent by weight of themedium is satisfactory. These carbon sources can be used individually,or several such sources may be combined in the medium.

Various nitrogen sources such as casein hydrolysates, amino acids, forexample, asparagine, glycine, arginine, digests of soybean meal, soybeanmeal, distillers solubles, and the like are readily assimilated by themethobottromycin and amethobottromycin producing microorganism and canbe used in fermentation mediums for the production of these antibiotics.In general, we find that organic sources of nitrogen, particularlysoybean meal, are very satisfactory for the production of the newantibiotics. The various organic and inorganic sources of nitrogen canbe used either alone or in combination in amounts ranging from about 0.2to about 6 percent by weight of the aqueous medium.

The following example illustrates a method of preparing the antibioticfrom which the molecule modifications of the present invention arederived, but it is to be understood that it is given for purposes ofillustration and not of limitation.

Preparation of Methobottromycin and Amethobottromycin A. Fermentation Amedium containing 1% dextrose, 0.3% meat extract, 1.0% tryptic digest ofcasein, and 0.5% sodium chloride was made up in water and adjusted to pH7.0 with sodium hydroxide sterilized and aseptically added to a slantculture of Streptomyces canadensis MA-959 (ATCC 17776) and the sporesscraped into suspension. About 3 ml. of this spore suspension wasaseptically added to a stoppered 2 liter bafflcd Erlenmeyer flaskcontaining 500 ml. of sterile aqueous medium consisting of 1% dextrose,0.3% meat extract, 1.0% tryptic digest of casin, and 0.5% sodiumchloride and the pH again adjusted to 7.0. The flask was incubated at28C. on a rotary shaker at a speed of 120 RPM with a 2-inch throw for aperiod of 48 hours.

This vegetative culture was then aseptically added to a SO-gallonstainless steel fermenter containing about 30 to 40 gallons of sterilemedium having a composition comprising 1.5% yeast autolysate, 1%dextrose, 0.25% sodium chloride with the pH adjusted to 7.5. Theinoculated medium was incubated at 28C. for 40 hours during which timeit was agitated with sterile air being passed through the medium at arate of about 3 cubic feet per minute. About 8.4 percent of thisvegetative culture was employed to inoculate a ISO-gallon stainlesssteel fermenter containing about 120 gallons of a medium having thecomposition comprising 1.5% yeast autolysate, 0.5% sodium chloride, and3% dextrose at pH 7.0 previously sterilized with steam at about 120C.for 15 minutes. The culture was incubated at 28C. with agitation andaeration at a rate of 10 CFM until maxium antibiotic yield was obtained.

B. Recovery The antibiotics of the present invention were recovered fromthe fermentation broth by adjusting the pH of the broth to 4.8 withhydrochloric acid and filtering. The filtered broth was passed through aDowex 50 X 2 sodium cycle resin (5 gallons) at a rate of 0.5 gallons perminute. The resin was washed with 10 gallons of water and eluted with 50gallons of methanol, 30% IN ammonia at a rate of 025 gallons per minute.Ten S-gallon cuts were taken and each was neutralized to pH 7 with 5%hydrochloric acid. The cuts were assayed and the active cuts wereevaporated to 7.5 gallons of water. The concentrate was adjusted to pH 8and extracted three times with an equal volume of chloroform and theextracts were dried over sodium sulfate. The rich chloroform was passedthrough a column containing a mixture of 70% Florisil, 30% Celite 545 ata 10 minute contact time. The absorption was followed with gallons ofchloroform wash followed by 50% chloroform-acetone elution at the samerate. V2 gallon cuts of the eluate were taken and the cuts were assayed.The active cuts were combined and evaporated to 0.5 gallons ofchloroform. The chloroform concentrate was dried to a syrup and taken upin about 160 ml. of methanol. Ten volumes of ethyl ether were added andthe insolubles filtered. A 1.2 N methanolic hydrochloric acid solutionwas added to the ether filtrate with stirring until no furtherprecipitation occured. The precipitate was filtered, washed with etherand dried.

Methobottromycin and amethobottromycin were isolated by partitionchromatography with 0.1 M pH 6.0 phosphate buffer as the eluent. Celiteimpregnated with capryl alcohol is used as the stationary phase. Thestationary phase is made by wetting 250 lbs. of acid washed celite(diatomaceous earth) with a solution of 6 gallons of capryl alcohol and24 gallons of acetone. The celite is air dried to remove acetone andpacked in o a :7! me n wherein M is des-carbomethoxy methobottromycin,descarbomethoxy amethobottromycin or mixtures thereof; R and R can bethe same or different hydro gen, alkyl radical, aryl radical or alkarylradical said alkyl groups containing 1 to about 12 carbon atoms. Areaction for the preparation of the molecular modifications of thepresent invention takes place by reacting methobottromycin andamethobottromycin with varia satisfactory column in thin layers withtamping, to inous amines is shown as follows:

sure uniformity. After packing, the column is washed with one-halfvolume of 0.1 M pH 6.0 phosphate buffer. About grams of the crudehydrochloride described above is dissolved in 8 gallons of pH 6.0 buffern-cou o c u-c-unn' 21-10 11' MCNRR' ca oir wherein M, R and R are thesame as above.

Another method for the preparation of the molecular modifications of thepresent invention is illustrated as follows:

and placed on the column. The column was developed wherein M, R-and Rare the same as above.

with pH 6.0 buffer taking 5 gallon cuts. Each fraction is assayed andexamined by paper chromatography. The rich fractions were worked up byconcentration to about one-fifth volume, adjusted to pH 8.5 andextracted two times with an equal volume of chloroform. The chloroformsolutions were evaporated to dryness. The residual solids were dissolvedin a small amount of methanol (about 500 ml.) and diluted with 20volumes of ethyl ether and filtered. To the filtrate was added 1.2 Nmethanolic hydrochloric acid until further addition caused noprecipitation.

The antibiotic hydrochlorides were collected and dried in vacuo with thefollowing results:

The following examples illustrate methods of preparing the antibioticmolecular modifications of the present invention. It is to beunderstood, however, that they are given for the purposes ofillustration and not of limitation.

EXAMPLE 1 Preparation of Methobottromycin Methylamide Methobottromycin(2.5 g.) is dissolved in 30 ml. of a methanolic solution containing 10percent by weight of methylamine. The mixture is sealed in a Cariustube, and heated at 50C. for 20 hours. The reaction mixture isevaporated to dryness and the last trace of methylamine is removed. Theresidue is crystallized from ethyl acetate to yield 1.9 g., m.p.225228C. dec. [(11, 23(C l, EtOH). Anal. Calcd. for C H N O S: C,

61.3; H,7.68; N, 15.33. Found: C, 61.0; H, 7.68; N, 15.39.

The hydrochloride is prepared by precipitation from ether by theaddition of methanolic hydrogen chloride. Anal. Calcd. for C., H N OS.HC1.2H O: C, 56.5; H, 7.7; N, 14.15. Found: C, 56.20; H, 7.6; N,14.00.

The infrared absorption spectrum of methobottromycin methylamideindicates the more significant of the characteristic peaks occur at thefollowing wave lengths expressed in reciprocal centimeters (cm): 1690,1662, 1632, 1620, 1510, 1450, 1428, 1372, 1356, 1332, 1278, 1255, 1232,1140, 1010, 711 and 700.

Less prominent but distinguishable bands are observed as follows: 1651,1530, 1495, 1410, 1308, 1221, 1208,1195,1170,1115,1l00, 768 and 755 cm.

EXAMPLE 2 Preparation of Amethobottromycin Methylamide 2.5 Grams ofamethobottromycin is dissolved in 30 ml. of a methanolic solutioncontaining 10 percent by weight of methylamine. The mixture is heated to50C. for 20 hours in a sealed tube. Amethobottromycin methylamide isrecovered and isolated according to the process described in Example 1.

EXAMPLE 3 Preparation of Mixture of Methobottromycin andAmethobottromycin Methylamide 2.5 Grams of a 70-30 percent mixture ofmethobottromycin and amethobottromycin is dissolved in 30 ml. of amethanolic solution containing 10 percent by weight of methylamide. Themixture is heated to 50C. for 20 hours in a sealed tube. The mixture ofmethobottromycin and amethobottromycin methylamide is recovered andisolated according to the process described in Example 1.

EXAMPLE 4 Preparation of Methobottromycin Ethylamide Methobottromycin(0.5 g.) is dissolved in 30 m1. of a methanolic solution containing 10percent by weight of ethylamine. The mixture is sealed in a Carius tube,and heated at 50C. for 20 hours. The reaction mixture is evaporated todryness. The residue is recrystallized from ethyl acetate to yieldmethobottromycin ethylamide, m.p. 174-180C. dec., [01],, 16(C 0.1, 95%EtOH). Anal. Calcd. for C,, H N O S.2H O: C, 59.20; H, 7.9; N, 14.41; S,3.7. Found: C, 59.20; H, 7.63; N, 14.1; S, 4.00.

EXAMPLE 5 Preparation of Amethobottromycin Ethylamide 0.5 Gram ofamethobottromycin is dissolved in 30 ml. of a methanolic solutioncontaining percent by weight of ethylamine. The mixture is heated to50C. for hours in a sealed tube. Amethobottromycin ethylamide isrecovered and isolated according to the procedure described in Example4.

EXAMPLE 6 Preparationof Mixture of Methobottromycin andAmethobottromycin n-propylamides 1.0 Gram of a 80-20 percent mixture ofmethobottromycin and amethobottromycin is dissolved in 30 ml. of amethanolic solution containing 10 percent by weight of n-propylamine.The mixture is heated to 50C. for 20 hours in a sealed tube. The mixtureof the methobottromycin and amethobottromycin npropylamides is recoveredand isolated according to the procedure described in Example 1.

EXAMPLE 7 Preparation of Methobottromycin B-phenylethylamide 1.0 Gram ofmethobottromycin is dissolved in 10 ml. of a methanolic solutioncontaining 20 percent by weight of B-phenylethylamine. The mixture isheated to 50C. for 20 hours in a sealed tube. MethobottromycinB-phenylethylamide is recovered and isolated according to the proceduredescribed in Example 1.

EXAMPLE 8 Preparation of Amethobottromycin 1-naphthyl methylamide 1.0Gram of amethobottromycin is dissolved in 20 ml. of a methanolicsolution containing 10 percent by weight of l-naphthylmethylamine. Themixture is heated to 50C. for 20 hours in a sealed tube.Amethobottromycin l-naphthylmethylamide is recovered and isolatedaccording to the procedure described in Example 1.

EXAMPLE 9 Preparation of Methobottromycin Cyclohexylamide 1.0 Gram ofmethobottromycin is dissolved in 10 ml. of a methanolic solutioncontaining 20 percent by weight of cyclohexylamine. The mixture isheated to 50C. for 20 hours in a sealed tube. Methobottromycincyclohexylamide is recovered and isolated according to the proceduredescribed in Example 1.

EXAMPLE 10 Preparation of Methobottromycin Benxylamide EXAMPLE 1 1Preparation of Methobottromycin 2,3Propandiol- 1 -Amide 1.0 Gram ofmethobottromycin is dissolved in 10 ml. of a methanolic solutioncontaining 20 percent by weight of 2,3-propandiol-lamine. The mixture isheated to 50C. for 20 hours in a sealed tube. Methobottromycin2,3-propandiol-l-amide is recovered and isolated according to theprocedure described in Example 1.

EXAMPLE 12 Preparation of Methobottromycin Ethanolamide 1.0 Gram ofmethobottromycin is dissolved in 10 ml. of a methanolic solutioncontaining 20 percent by weight of ethanolamine. The mixture is heatedto 50C. for 20 hours in a sealed tube. Methobottromycin ethanolamide isrecovered and isolated according to the procedure described in Example1.

EXAMPLE 13 Preparation of t-butylamide of Methobottromycin A solution of2.0 grams of methobottromycin dissolved in 100 ml. of acetone and 100ml. of 0.1 N sodium hydroxide is stirred at C. for 3 hours. Hydrochloricacid is added to adjust the pH to 6.3 and the solution concentrated toabout 50 ml. and allowed to cool. The free acid methobottromycincrystallizes on cooling and is collected by filtration and washed withwater. After drying at 80C. at 1 mm for 1 hour, 1.85 grams of the freeacid of methobottromycin is obtained, m.p. 195-200C.

450 Milligrams of the free acid is dissolved in ml. of dimethylformamideand 219 mg. of bis-(2,4-dinitrophenyl) carbonate is added. 0.14Milliliters of triethylamine is then added and the mixture is stirred at0C. for 45 minutes and then at room temperature for minutes. To thismixture is added 2 ml. of tbutylamine. The mixture is heated at 50-60C.for 30 minutes, diluted to about 25 ml. with chloroform and filtered.The filtrate is concentrated to near dryness under reduced pressure. Theresidue is dissolved in chloroform and then washed with saturated sodiumchloride solution containing 5 percent ammonia until the aqueous layeris colorless. The organic layer is dried over sodium sulfate and thenconcentrated to 390 mg. of a yellow glass. Preparative tlc yields 197mg. of the t-butylamide of methobottromycin. Anal. Calcd. for C., H N OS: C, 62.54; H, 8.05; N, 14.59; S, 3.17. Found: C, 62.33; H, 8.11; N,14.35; S, 3.61. The infrared absorption spectrum of methobottromycintbutylamide indicates that the more significant of the characteristicpeaks occur at the following wave lengths expressed in reciprocalcentimeters (cm): 1655, 1648, 1530, 1495, 1460. 1388, 1374, 1362, 1305,1255, 1220, 1140, 1115, 718 and 700. Less prominent but distinguishablebands are observed as follows: 1508, 1340, 1195, 1178 and 752 cm.

EXAMPLE 14 Preparation of Aniline Amide of Methobottromycin 8OMilligrams of the free acid of methobottromycin is dissolved in 2 ml. ofdimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonateis added. 10 Milligrams of triethylamine is then added and the mixtureis allowed to stand overnight. To this mixture 10 ml. of methylenechloride is added, the mixture is then washed with water, and the waterwashes discarded. The methylene chloride is evaporated and the anilineamide of methobottromycin is recovered in a yield of about 70 percent.

EXAMPLE Preparation of p fluoro Aniline Amide of Methobottromycin 80Milligrams of the free acid of methobottromycin is dissolved in 2 ml. ofdimethylformamide and 40 milligrams of bis-2,4-dinitriphenyl carbonateis added. 10 Milligrams of triethylamine is then added and the mixtureis allowed to stand for 30 minutes. 10 Milligrams of p-fluoro aniline isthen added and the mixture is allowed to stand overnight. To thismixture 10 ml.-of methylene chloride is added, the mixture is thenwashed with water and the water washes discarded. The methylene chlorideis evaporated and the p-fluoro aniline amide of methobottromycin isrecovered in a yield of about 60 percent.

EXAMPLE 16 Preparation of N,N-dimethyl Amide of MethobottromycinMilligrams of the free acid of methobottromycin is dissolved in 2 ml. ofdimethylformamide and 40 milligrams of bis-2,4-dinitropheny] carbonateis added. 10 Milligrams of triethylamine is then added and the mixtureis allowed to stand for 30 minutes. 10 Milligrams of dimethylamine isthen added and the mixture is allowed to stand overnight. To thismixture 10 ml. of methylene chloride is added, the mixture is thenwashed with water and the water washes discarded. The methylene chlorideis evaporated and the N,N- dimethyl amide of methobottromycin isrecovered in a yield of about 56 percent.

EXAMPLE 17 Preparation of 2-aminobenzimidazole Amide ofAmethobottromycin 1.0 Gram of amethobottromycin is dissolved in 50 ml.of acetone and 50 ml. of 0.1 N sodium hydroxide is added and let standfor 2 hours. Hydrochloric acid is added to adjust to pH 6.2 and thesolution is concentrated to one-half volume and allowed to cool. About0.8 grams of the free acid of amethobottromycin crystallizes from thissolution. 80 Milligrams of the free acid of amethobottromycin isdissolved in 2 ml. of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamine is thenadded and the mixture is allowed to stand for 30 minutes. 10 Milligramsof 2- aminobenzimidazole is then added and the mixture is allowed tostand overnight. To this mixture 10 ml. of methylene chloride is added,the mixture is then washed with water and the water washes discarded.The methylene chloride is evaporated and the 2- aminobenzimidazole amideof amethobottromycin is recovered in a yield of about 50 percent.

EXAMPLE 18 Preparation of Morpholine Amide of Methobottromycin 80Milligrams of the free acid of methobottromycin is dissolved in 2 ml. ofdimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonateis added. 10 Milligrams of triethylamine is then added and the mixtureis allowed to stand for 30 minutes. 10 Milligrams of morpholine is thenadded and the mixture is allowed to stand overnight. To this mixture 10ml. of methylene chloride is added, the mixture is then washed withwater, and the water washes discarded. The methylene chloride isevaporated and the morpholine amide of methobottromycin is recovered ina yield of about 65 percent.

EXAMPLE 19 Preparation of N,N-dimethylamino Ethyl Amide ofMethobottromycin 80 Milligrams of the free acid of methobottromycin isdissolved in 2 ml. of dimethylformamide and 40 milligrams ofbis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamineis then added and the mixture is allowed to stand for 30 minutes. 10Milligrams of N,N-dimethylamino ethylamine is then added and the mixtureis allowed to stand overnight. To this mixture, l ml. of methylenechloride is added, the mixture is then washed with water, and the waterwashes discarded. The methylene chloride is evaporated and theN,N-dimethylamino ethyl amide of methobottromycin is recovered in ayield of about 60 percent.

EXAMPLE 2O Preparation of Methyl Ester of Phenylalanine Amide ofMethobottromycin 8O Milligrams of the free acid of methobottromycin isdissolved in 2 ml. of dimethylformamide and 40 milligrams ofbis-2,4-dinitrophenyl carbonate is added. 10 Milligrams of triethylamineis then added and the mixture is allowed to stand for 30 minutes. 10Milligrams of methyl ester of phenylalanine is then added and themixture is allowed to stand overnight. To this mixture, 10 ml. ofmethylene chloride is added, the mixture is then washed with water, andthe water washes discarded. The methylene chloride is evaporated and themethyl ester of phenylalanine amide of methobottromycin is recovered ina yield of about 58 percent.

EXAMPLE 21 Preparation of Dodecyl Amide of Methobottromycin 80Milligrams of the free acid of methobottromycin is dissolved in 2 ml. ofdimethylformamide and 40 milligrams of bis-2,4-dinitrophenyl carbonateis added. 10 Milligrams of triethylamine is then added and the mixtureis allowed to stand for 30 minutes. l0 Milligrams of dodecylamine isthen added and the mixture is allowed to stand overnight. To thismixture 10 ml. of methylene chloride is added, the mixture is thenwashed with water and the water washes discarded. The methylene chlorideis evaporated and the dodecyl amide of methobottromycin is recovered ina yield of about 62 percent.

EXAMPLE 22 Preparation of Imidazole Amide of Amethobottromycin 80Milligrams of the free acid of amethobottromycin is dissolved in 2 ml.of dimethylformamide and 40 milligrams of bis-2,4-dinitrophenylcarbonate is added. 10 Milligrams of triethylamine is then added and themixture is allowed to stand for 30 minutes. 10 Milligrams of imidazoleis then added and the mixture is allowed to stand overnight. To thismixture 10 ml.'of methylene chloride is added, the mixture is thenwashed with water and the water washes discarded. The methylene chlorideis evaporated and the imidazole amide of amethobottromycin is recoveredin a yield of about 59 percent.

The molecular modification amides of amethobottromycin,methobottromycin, and mixtures thereof are valuable antibacterial agentswhich, as has been pointed out above, are active in inhibiting thegrowth of various gram positive organisms. However, these, antibioticsare extremely useful in the treatment of chronic respiratory disease ofchickens and infectioussinusitis of turkeys. In this embodiment of thepresent invention it has been found that chronic respiratory disease ofchickens and infectious sinusitis of turkeys may be effectivelyinhibited by the use of molecular modification amides of the presentinvention which may be administered by either the subcutaneous or theoral route. Furthermore, it has been found that the molecularmodification amides of the present invention are effective incontrolling chronic respiratory disease in chickens and infectioussinusitis in turkeys when administered in dosages ranging from about 0.1mg./kg. to about 250 mg./kg. of body weight ofthe bird without theresulting danger of toxicity and preferably from 0.5 mg./kg. to I50mg./kg. of body weight of the birds, depending on the route of therapy.

In order to illustrate the activity and the antibiotic advantages of thepresent invention in utilizing the molecular modification amides ofamethobottromycin, methobottromycin, and mixtures thereof, the followingtest examples are given. It is understood, however, that they are givenmerely for the purpose of illustration and in no way are they to betaken as limiting.

In order to illustrate the activity of the compounds of the presentinvention as antibiotic agents against chronic respiratory disease ofchickens and infectious sinusitis of turkeys the following examples aregiven:

EXAMPLE 23 Activity of the Methyl Amide of Methobottromycin againstMycoplasma gallisepticum (PPLO) Infections in Chickens (OralAdministration) of Non- Avg. Wt. Infected Mortality Treatment Gain-gmBody Wt. Gain Dead/Total Non-Infected Control 212 0/6 Infected Control139 67 10/21 Methyl Amide of Methobottromycin 25 mgJkg. I73 82 0/6 50mgJkg. 208 98 3/6 I00 mgJkg. I69 0/6 In the above test the body weightgain in the birds treated by the method of the present invention showeda significant increase over those of the infected controls. In addition,where 11 out of 21 or 52 percent of the infected controls died, only 3out of 18 or 17 percent of the birds treated by the method of thepresent invention died.

EXAMPLE 24 Activity of the Methyl amide of Methobottromycin againstMycoplasma gallisepticum (PPLO) Infections in Chickens (SubcutaneousAdministration) of Non- Avg.Wt. Infected Mortality Treatment Gain-gm.Body Wt. Gain Dead/Total Non-Infected Control 216 /6 Infected Control107 50 7/18 Methyl Amide of Methobottromycin 0.5 mgJkg. 157 73 0/6 1.0mgJkg. 193 89 2/6 2.0 mgJkg. 196 91 2/6 Weeks-n .9 2 .9. 9L9.

In the above test the body weight gain in the birds treated by themethod of the present invention showed a significant increase over thoseof the infected controls. In addition, were 7 out of 18 or 39 percent ofthe infected controls died, only 4 out of 24 or 17 percent of the birdstreated by the method of the present invention died.

To further illustrate the antibiotic activity of the amide compositionsof the present invention, these compositions were tested against PPLOmicroorganisms. The results of in vitro assays (agar diffusion method)are illustrated but not limited to the following example:

Example 25 Amide in Vitro Activity Zone of Inhibition: mm 250 'yglmlAmide seo 'yg/ml 1.0 mglml Benzyl Amide of Methobottromycin AnilineAmide of Methobottromycin p-Fluoroaniline Amide of MethobottromycinMorpholine Amide of Methobottromycin Methyl B-phenylalanine amide ofMethobottromycin Dodecyl Amide of Methobottromycin lmidazole Amide ofMethobottromycin EXAMPLE 26 In Vivo Activity of Methobottromycin andAmethobottromycin Amides against Mycoplasma gallisepticum in ChickensGroups of 5-day old white Leghorn pullets were infected via the air sacroute with a broth culture of Mycoplasma gallisepticum. Various amidesof the present invention were administered in a water solution by thesubcutaneous route with dosages given at 2 and 18 hours after infectionin the amount of 10, 25, and 50 mgjkg. of body weight for each group ofpullets. The birds were observed for development of symptoms of chronicrespiratory disease, and the results were as follows:

Amides Results at Test Termination Disease evident Disease arrestedDisease arrested Disease arrested Disease arrested Disease arrestedUntreated Controls Methylamide Ethanolamide 2,3-propandiolamidep-Fluoroaniline amide Pyrolidineamide It is evident from the test dataabove that chickens that were infected with Mycoplasma gallisepticum hadthe infectious chronic respiratory disease arrested when treated with aslittle as 10 mgjkg. of the amides of the present invention. Whereas, theuntreated chickens showed continued infection of chronic respiratorydisease.

protect 50% of the infected mice.

The in vivo test, the results of which are listed above,

was carried out by giving the mice a two-dose treatment at O and 6 hoursafter infection and the test was concluded seven days after infection.

While specific embodiments of the present invention have been named anddescribed, it will be apparent to those skilled in the art that changesmay be made in the detail shown without departing from the spirit of thepresent invention or the scope intended. Any departure from the abovedescription which conforms to the present invention is intended to beincluded within the scope 'of the claims.

What is claimed is:

1. Methobottromycin methylamide being characterized by the followingproperties:

a. melting at 225228C. with decomposition,

b. having a specific rotation of [07],, 23 (C l,

percent ethyl alcohol),

0. having the empirical formula C H N O S,

(1. containing 61.3% carbon, 7.68% hydrogen and 15.33% nitrogen, and

e. having an infrared absorption spectrum exhibiting characteristicpeaks at the following wave lengths expressed in reciprocal centimeters:1690, 1662, 1632,1620,1510,1450,1428,1372,1356,1332, 1278, 1255, 1232,1140, 1010, 711 and 700.

2. Methobottromycin t-butylamide being characterized by the followingproperties:

a. having the empirical formula C H N O S,

b. containing 62.54% carbon, 805% hydrogen, 0

14.59% nitrogen and 3.17% sulfur, and 0. having an infrared absorptionspectrum exhibiting the following characteristic peaks expressed in re-

2. Methobottromycin t-butylamide being characterized by the followingproperties: a. having the empirical formula C45H69N9O6S, b. containing62.54% carbon, 8.05% hydrogen, 14.59% nitrogen and 3.17% sulfur, and c.having an infrared absorption spectrum exhibiting the followingcharacteristic peaks expressed in reciprocal centimeters: 1655, 1648,1530, 1495, 1460, 1388, 1374, 1362, 1305, 1255, 1220, 1140, 1115, 718and
 700. 3. A method of treating poultry infected withpleuropneumonia-like organisms which comprises administering to saidinfected poultry an effective dose of methobottromycin methylamide ormethobottromycin t-butylamide.
 4. The method of claim 3 wherein saideffective dose is in the range of from about 0.1 mg./kg. to about 250mg./kg. of body weight of said infected poultry. 700.